title: Heparan sulfate mediates trastuzumab effect in breast cancer cells.
abstract: BACKGROUND
  Trastuzumab is an antibody widely used in the treatment of breast cancer cases that test positive for the human epidermal growth factor receptor 2 (HER2). Many patients, however, become resistant to this antibody, whose resistance has become a major focus in breast cancer research. But despite this interest, there are still no reliable markers that can be used to identify resistant patients. A possible role of several extracellular matrix (ECM) components--heparan sulfate (HS), Syn-1(Syndecan-1) and heparanase (HPSE1)--in light of the influence of ECM alterations on the action of several compounds on the cells and cancer development, was therefore investigated in breast cancer cell resistance to trastuzumab.
  
  
  METHODS
  The cDNA of the enzyme responsible for cleaving HS chains from proteoglycans, HPSE1, was cloned in the pEGFP-N1 plasmid and transfected into a breast cancer cell lineage. We evaluated cell viability after trastuzumab treatment using different breast cancer cell lines. Trastuzumab and HS interaction was investigated by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). The profile of sulfated glycosaminoglycans was also investigated by [35S]-sulfate incorporation. Quantitative RT-PCR and immunofluorescence were used to evaluate HPSE1, HER2 and Syn-1 mRNA expression. HPSE1 enzymatic activity was performed using biotinylated heparan sulfate.
  
  
  RESULTS
  Breast cancer cell lines responsive to trastuzumab present higher amounts of HER2, Syn-1 and HS on the cell surface, but lower levels of secreted HS. Trastuzumab and HS interaction was proven by FRET analysis. The addition of anti-HS to the cells or heparin to the culture medium induced resistance to trastuzumab in breast cancer cells previously sensitive to this monoclonal antibody. Breast cancer cells transfected with HPSE1 became resistant to trastuzumab, showing lower levels of HER2, Syn-1 and HS on the cell surface. In addition, HS shedding was increased significantly in these resistant cells.
  
  
  CONCLUSIONS
  Trastuzumab action is dependent on the availability of heparan sulfate on the surface of breast cancer cells. Furthermore, our data suggest that high levels of heparan sulfate shed to the medium are able to capture trastuzumab, blocking the antibody action mediated by HER2. In addition to HER2 levels, heparan sulfate synthesis and shedding determine breast cancer cell susceptibility to trastuzumab.
authors.affiliation: Department of Biochemistry, Universidade Federal de São Paulo, Rua Três de Maio, 100, Vila Clementino, 04044-020, São Paulo, SP, Brazil. eloahrabello@yahoo.com.br.
authors.forename: Eloah Rabello
authors.initials: ER
authors.lastname: Suarez
authors.forename: Edgar Julian
authors.initials: EJ
authors.lastname: Paredes-Gamero
authors.forename: Auro
authors.initials: A
authors.lastname: Del Giglio
authors.forename: Ivarne Luis dos Santos
authors.initials: IL
authors.lastname: Tersariol
authors.forename: Helena Bonciani
authors.initials: HB
authors.lastname: Nader
authors.forename: Maria Aparecida Silva
authors.initials: MA
authors.lastname: Pinhal
mesh_terms: D061067:Antibodies, Monoclonal, Humanized / Q000378:metabolism / Q000494:pharmacology*; D000970:Antineoplastic Agents / Q000378:metabolism / Q000494:pharmacology*; D001943:Breast Neoplasms / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D019008:Drug Resistance, Neoplasm; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D006025:Glycosaminoglycans / Q000378:metabolism; D006497:Heparan Sulfate / Q000378:metabolism*; D006801:Humans; D061986:MCF-7 Cells; D011485:Protein Binding; D021381:Protein Transport; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D053668:Syndecan-1 / Q000235:genetics / Q000378:metabolism; D000068878:Trastuzumab; D000099175:Heparanase
chemical_list: D061067:Antibodies, Monoclonal, Humanized; D000970:Antineoplastic Agents; D005966:Glucuronidase; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D018719:Erb-b2 Receptor Tyrosine Kinases; D053668:Syndecan-1; D000068878:Trastuzumab; D000099175:Heparanase; C013786:A73025